Project Summary Drug abuse induces and facilitates dopamine neurotransmission through the mesolimbic dopaminergic pathway, which modifies reward-related behaviors and is associated with the development of drug addiction. Mounting evidence suggests that addicted individuals exhibited significant deficits on the level and signaling of dopamine D2 receptor (D2R), which contribute to decreased sensitivity of addicted subjects to natural reinforcers and predispose them to more potent drug stimulation of euphoria. Therefore, D2R presents a highly promising therapeutic target for drug abuse, as supported by both pre-clinical and clinical studies. Although D2R agonists are being intensively studied as therapeutic intervention for drug addiction, their success have been greatly hampered due to poor adherence and efficacy, or associated side effects related to dopaminergic medications. Structural information on D2R will not only reveal its signaling mechanisms, but also provide required information for rational drug design. As a member in the G protein-coupled receptor (GPCR) family, D2R is notorious to form diffraction-quality crystals that are essential for the determination of high-resolution structures by X-ray crystallography. This grant proposal aims to develop a comprehensive and robust platform for structural and functional studies of D2R. To achieve this goal, we will address three major aims: 1) develop optimized protocols for obtaining purified receptors in quantities sufficient for crystallization attempts and functional studies, 2) use stable receptors from Aim 1 to screen for optimal ligands and assess the quality of receptor-ligand complexes with an array of quality- control analyses, and 3) co-crystallize receptor-ligand complexes for structure determination and set up receptor- containing nanodiscs for functional studies as well as searching for high-affinity nanobodies or compounds for D2R. This platform will involve multiple steps that are closely interconnected and looped through a forward and backward feedbacks system. Although highly challenging in nature, we have acquired a few human D2R constructs that could be expressed and purified to more than 1 mg protein per liter of biomass in our laboratory, which is essential for the accomplishment of subsequent aims. The significance of this study is multi-fold on dopamine receptor family and related drug abuse studies: 1) gain insights into biased agonism, 2) reveal information on allosteric modulations, 3) screen extensively to produce new high-affinity ligands for D2R, 4) characterize the mechanisms of D2R signaling and ligand selection between different dopamine receptor subfamilies, 5) stimulate structure-based drug design, 6) establish the platform for exploring the polypharmacology of the ligands that target D2R, and 7) pave the road for GPCR/G protein complex signaling studies and understanding the activation mechanism in the future.